Annastella Gambini

Eukaryotic N10-formyl-H4folate:Methionyl-tRNAf transformylase: Some properties of the Euglena gracilis enzyme

Abstract The N 10 - formyl-H 4 folate:methionyl-tRNA fMet transformylase was extracted from Euglena cells, and the level of enzyme activity found to be of 0.15 pmol/min per 106 cells for autotrophic and 0.016 pmol/min per 106 cells for streptomycin-bleached cells. The enzyme has been purified by DEAE-cellulose and CM-Sephadex chromatography about 1000-fold from autotrophic cells and about 650-fold from chloroplasts obtained by the non-aqueous technique. A methionyl-tRNA synthetase with apparently high specificity for the initiator tRNAMet has also been found which allowed the estimation of enzyme activity even with unfractionated tRNAs from different sources, since only formylatable species were present in methionylated form. As judged from the initial velocity the Euglena transformylase showed the same kinetic behaviour towards Met-tRNAfMet from E. gracilis, Escherichia coli and yeast. The chloroplast enzyme showed different properties from those of the bacterial enzyme. It appears to have a partial requirement for K+, a higher molecular weight, and a lower Mg2+ optimum. Moreover, the chloroplast transformylase showed sigmoidal rather than hyperbolic kinetics respect to Met-tRNAfMet ( n H = 1.81 ; K′ = 4 · 10 −14 ) as well as N 10 - formyl-H 4 folate ( n H = 2.46 ; K′ = 5.2 · 10 −16 ), thus indicating a strong positive cooperativity for both substrates.

Repression of folate synthesis in the logarithmic phase of Euglena gracilis growth

Abstract Microbiological assay showed that in Euglena gracilis cultures the amount of cell folates reaches its maximum at the beginning of the culture cycle and rapidly and markedly decreases long before the cells reduce their duplication rate. [ 3 H]Folic acid was a suitable precursor of Euglena folates (a full recovery of growth in sulfanilamide inhibited cultures was obtained by addition of folic acid), and a complete radiochromatographic profile of cell folates was obtained by separation on G-25 columns. This allowed the measurement of the rate of folate degradation, obtained from the rate of radioactivity disappearance in chromatographic patterns of extracts corresponding to increasing times of culture cycles. With the exception of the stationary phase, the process of folate degradation showed a first order kinetics with a rate constant of 5.4 + 10 −4 min −1 and a half-life of 21 h and 12 min. The rate of folate biosynthesis was calculated by adding the amount of degradation to the measured increase (or decrease) in cell folates. The specific rate reached its maximum (8.6 ng of folinic acid equivalent h −1 for 10 6 cells) as the culture entered the logarithmic phase of growth and rapidly decreased to about 1/20 of this value before leaving it. This indicates that the logarithmic phase of growth corresponds to a phase in which folate biosynthesis is strongly repressed.

Culture cycle dependence of folate interconversion and related enzymes in Euglena gracilis

Abstract Folates are involved in one-carbon metabolism in which one-carbon groups of increasing reduction state (formyl, methylene and methyl) are cyclically accepted and donated by the coenzyme form of folic acid, tetrahydrofolic acid. The latter originates by reduction of dihydrofolic acid, the coenzymatically inactive form. Euglena culture cycle dependence of folate distribution in oxidized, formyl and methyl forms and of enzyme activities for folate interconversion were studied. Distribution levels of all the components examined varied widely during the culture, and many of these changes occurred in the logarithmic phase of growth. In the phase of folate synthesis, there was an appreciable delay in the conversion of oxidized to reduced forms and of formyl to methyl forms. This delay appeared to be correlated with the level of corresponding enzymes. The methyl folate peak coincided with the highest level of total cell folates, at which point a severe repression of folate synthesis began. During the last phase of exponential growth, when cell folate content was reduced to one-fifth and folates had shorter glutamate chains, the level of coenzymatically inactive and inhibitory oxidized forms increased again. The reduced efficiency of the system and the change in growth rate are discussed. The activity patterns of dihydrofolate reductase and methylene tetrahydrofolate reductase were markedly different. The peak in methylene tetrahydrofolate reductase activity coincided with the absence of oxidized folates. A regulation of folate synthesis by the level of methyl folates and of methylene tetrahydrofolate reductase synthesis by the level of oxidized forms is proposed.

Inhibitor studies of methionyl-tRNA transformylase of Euglena gracilis

Inhibitor studies of the only known eukaryotic methionyl-tRNA transformylase (10-formyltetrahydrofolate:L-methionyl-tRNA N-transformylase, EC 2.1.2.9) were carried out. All the natural pteroylglutamic acid derivatives examined, with the exception of pteroylglutamic acid, are inhibitors. The most effective is 5-methyltetrahydrofolate (5-CH3-H4PteGlu) (KI = 3 . 10(-6) M), which is the only noncompetitive inhibitor of the enzyme. All the other derivatives tested are competitive, and H4PteGlu shows a cooperative inhibition. These and other data obtained with pteroylglutamic analogues show that, in contrast to the bacterial enzyme, Euglena transformylase is also inhibited by compounds without a fully reduced pyrazine ring and is very sensitive to compounds with a methyl group in position 5 or 10 of the pteridine ring.

An online Biology course: a teaching-learning experiment

We have been experimenting with an online course in «teaching Biology» for students of the Primary Education Degree course1 for about two years. This learning activity was created for students who work or live far from Milan and cannot regularly attend the University. Lessons are based on the participant’s personal experience of the relationship with living organisms such as plants, animals, etc. This is the basis for the professional training of primary school teachers who will teach science-related subjects to children. Students who cannot attend lessons have to work very hard to carry out practical activities in Biology-related contents. During this Distance-Learning course they are helped to follow it and, finally, to produce materials that will be evaluated during the examination. Students can discuss their learning with peers, with teacher and with tutor using a forum cafe, and they have five thematic forums to carry out reflections, to ask for explanations, etc. These interactions constitute the real advantage of this type of teaching-learning mode, which encourages collaborative learning and the sharing of experiences and of acquired knowledge. The role of the teacher goes beyond the traditional view: first, it consists of planning activities; this is followed by monitoring interactions, in correcting Biology contents, in suggesting further studies, etc., while students are performing their tasks. The site is still in an experimental phase. At present, only a few students can participate, but three or four editions of the online course are provided during a year.

Quality indicators for the analysis of communication in an online course

This study describes the development and validation of quality indicators for analyzing forums interactions in an online course in biology teaching. The aim is to evaluate the quality of communication so as to strengthen the tutor’s role and help students learn fundamental biology concepts while enhancing their collaboration competencies. The indicators are used to analyze cognitive, metacognitive and relational aspects, drawing on a content analysis methodology. The model appears to have a wide range of possible applications in other online courses.